1a.Objectives (from AD-416):
The primary objective of this project is to determine the effects of plant genotype, rhizobia genotype and plant x rhizobia interaction effects on biomass accumulation and nitrogen fixation in peas, lentils, and chickpeas. Secondary objectives include developing a regional collection of microbes that fix nitrogen in peas, lentils, and chickpeas cultivated in the U.S. Pacific Northwest; detecting genetic variation among isolates of Rhizobium leguminosarum and Mesorhizobium ciceri and determining population structure of these micobes both within and between production fields in WA, and examining how various soilborne pathogens of pulse crops affect the formation of root nodules, the amount of rhizobia present in nodules, and the efficiency of nitrogen fixation in infected pulse crops. Documents SCA with WSU.

1b.Approach (from AD-416):
Strains of R. leguminosarum will be isolated from nodules of various lentil and pea cultivars grown in several different locations in WA. Strains of M. ciceri will be similary isolated from nodules of several chickpea cultivars. Genomic DNA will be extracted from the isolates. Genetically distinct isolates of R. leguminosarum and M. ciceri will be identified by using polymerase chain reaction (PCR) to amplify 16S rDNA, followed by sequence analysis. Genetically distinct isolates will be stored at -80 C as glycerol stocks. A subsample of at least five distinct isolates of R. leguminosarum and five distinct isolates of M. ciceri will be used to examine rhizobia genotype effects on biomass and nitrogen fixation in peas, lentils, and chickpeas.

Five different genotypes each of commercial cultivars of peas, lentils, and chickpeas, will be inoculated with each isolate of R. leguminosarum or M. ciceri. Replicated plants will be grown individually in sterile “conetainers” using a low-nutrient sand-based potting mix. Each conetainer will be fertilized with 10 ml 2 M ammonium sulfate with 10% isotopic 15-N at 1 wk and 2 wk to isotopically “label” the plant biomass. At 3 wk and 6 wk after planting, each conetainer will receive 5 ml total volume of a broth culture of a single rhizobium isolate at 105 – 106 cells per ml. Plants will be harvested at 12 wk after planting. The number of nodules per plant will be determined. Biomass will be dried, weighed, ground, and analyzed for both total N and 15-N. The proportion and total amount of plant N that was fixed by the rhizobial symbiont will be determined with this information.

Real-time (TaqMan) PCR assays will be developed for quantifying the amount of R. leguminosarum or M. ciceri present in root nodules. Briefly, isolates will be amplified using SRAP markers to identify amplicons that are present in all examined isolates of R . leguminosarum or M. ciceri. Species-specific amplicons will be cloned and sequenced and the sequence will be analyzed using Primer Express software to identify candidate Taqman probes and primers.

Pea and lentil plants will be inoculated with R. leguminosarum and grown in greenhouse flats. Approximately 5 d after emergence plants will be inoculated with a zoospore suspension of the soilborne pathogen Aphanomyces euteiches. Control plants will comprise two treatments, one in which the plants are inoculated with only R. leguminosarum and the other being plants inoculated only with A. euteiches. Plants will be scored for disease severity 14 d after inoculation using a scale of 1 (healthy plant) to 5 (dead plant) that is well accepted for scoring Aphanomyces root rot in pea and lentil. The number of nodules per plant will be determined. DNA will be extracted from the entire root system of each plant. The amount of R. leguminosarum and A. euteiches DNA present in the root system will be determined using real-time PCR. Correlations between the amount of pathogen DNA and R. leguminosarum DNA present in root systems will be calculated, as will correlations between the number of nodules in root systems and the amount of A. euteiches and R. leguminosarum DNA present in roots.

3.Progress Report:

A total of 95 isolates of Rhizobium leguminosarum and 48 isolates of Mesorhizobium ciceri were isolated from pea and chickpea roots, respectively, from several locations in the US Pacific Northwest. Genetic relationships between isolates have been determined based on the differences in DNA sequences observed between isolates for several different genes, including nodC, nifH, recA, atpD, and 16S rRNA. This information will help researchers choose small sets of isolates for future studies that reflect the genetic diversity present in the entire collection. The results of this project relate to Subobjective 1A (Develop pea, lentil and chickpea cultivars with broad adaptation to diverse production environments and with resistance to fungal and viral pathogens) of the in-house associated project.